Mannich reaction-based combinatorial libraries identify antioxidant ionizable lipids for mRNA delivery with reduced immunogenicity

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Mannich reaction-based combinatorial libraries identify antioxidant ionizable lipids for mRNA delivery with reduced immunogenicity

Data availability

The data that support the findings of this study are available within the paper and its Supplementary Information files. Source data are provided with this paper.

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Acknowledgements

M.J.M. acknowledges support from a US National Institutes of Health (NIH) Director’s New Innovator Award (DP2 TR002776), a Burroughs Wellcome Fund Career Award at the Scientific Interface (CASI), a US National Science Foundation CAREER Award (CBET-2145491) and the American Cancer Society (number RSG-22-122-01-ET). E.L.H. acknowledges support from a US National Science Foundation Graduate Research Fellowship (DGE 1845298). C.G.F.-E. acknowledges support from a US National Science Foundation Graduate Research Fellowship (DGE 1845298), a GEM Fellowship and the NIH/National Cancer Institute Pre-doc to Post-doc Transition Award (F99 CA284294).

Author information

Author notes

  1. These authors contributed equally: Ningqiang Gong, Dongyoon Kim.

Authors and Affiliations

  1. Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA

    Ningqiang Gong, Dongyoon Kim, Emily L. Han, Rohan Palanki, Qiangqiang Shi, Xuexiang Han, Lulu Xue, Junchao Xu, Christian G. Figueroa-Espada & Michael J. Mitchell

  2. Department of General Surgery, The First Affiliated Hospital of USTC, Division of Life Science and Medicine, University of Science and Technology of China, Hefei, China

    Ningqiang Gong, Zilin Meng & Tianyu Luo

  3. Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Mohamad-Gabriel Alameh, Rakan El-Mayta, Garima Dwivedi, Drew Weissman & Michael J. Mitchell

  4. Penn Institute for RNA Innovation, University of Pennsylvania, Philadelphia, PA, USA

    Mohamad-Gabriel Alameh, Rakan El-Mayta, Garima Dwivedi, Drew Weissman & Michael J. Mitchell

  5. Department of Chemistry, Tsinghua University, Beijing, China

    Jinghong Li

  6. Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

  7. Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

  8. Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

  9. Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

Authors

  1. Ningqiang Gong
  2. Dongyoon Kim
  3. Mohamad-Gabriel Alameh
  4. Rakan El-Mayta
  5. Emily L. Han
  6. Garima Dwivedi
  7. Rohan Palanki
  8. Qiangqiang Shi
  9. Xuexiang Han
  10. Lulu Xue
  11. Junchao Xu
  12. Zilin Meng
  13. Tianyu Luo
  14. Christian G. Figueroa-Espada
  15. Drew Weissman
  16. Jinghong Li
  17. Michael J. Mitchell

Contributions

N.G., D.K. and M.J.M. conceived of and designed the experiments. N.G., D.K., M.-G.A., R.E.-M., G.D., Q.S., X.H., L.X., J.X., R.P., Z.M., T.L. and C.G.F.-E. performed the experiments. N.G., D.K. and R.E.-M. analysed the data. D.W. and J.L. involved in results discussion. N.G., D.K., E.L.H., R.E.-M. and M.J.M. wrote and edited the paper. M.J.M. supervised the entire project. All authors discussed the results and commented on the paper.

Corresponding author

Correspondence to Michael J. Mitchell.

Ethics declarations

Competing interests

N.G. and M.J.M. have filed a patent application related to this study. The other authors declare no competing interests.

Peer review

Peer review information

Nature Biomedical Engineering thanks Bruno De Geest, Shuai Liu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Evaluation of LNP-induced innate immunity activation in vivo using complete blood counting assay and ELISA assay.

C-a16, C-a16-Q, DLin-MC3-DMA LNPs or PBS was intravenously injected, and a complete blood counting (CBC) assay was performed 24 hours post-LNP injection. a, The levels of white blood cells (WBC), b, neutrophils (Neu), c, lymphocytes (Lym), and d, monocytes (Mon), in the blood were measured (n = 4). ELISA assay was used to quantify the levels of IL-2 (e) and TNF-α (f) in the mouse serum (n = 4). Statistical differences were calculated using one-way ANOVA with Tukey’s post hoc test.

Source data

Extended Data Fig. 2 C-a16 LNPs induce antigen-specific immune responses.

a–d, Quantification of LNP distribution in immune cell populations, including DCs (a), macrophages (b), B cells (c), and T cells (d), assessed by flow cytometry. Data are presented as mean ± SD (n = 5), and statistical differences were determined by one-way ANOVA. e, Immunofluorescence images of lymph nodes in mice after intramuscular injection of DiR dye-labeled LNPs. Nuclei are represented in blue, CD11c+ DCs in green, and DiR-labeled LNPs in red. f, Evaluation of OVA-specific T cell generation following two doses of C-a16-mOVA vaccines. Seven days after the boost dose, blood T cells were stained with SIINFEKL-H2Kb tetramer-APC antibody. After that, SIINFEKL-H2Kb tetramer+ cells were detected by flow cytometry (f) and quantified (g). h and i, Investigation of vaccine-induced immune responses’ ability to eliminate SIINFEKL epitope+ target cells. A mixture of CFSElow SIINFEKL-loaded and CFSEhigh non-loaded splenocytes (in a 1:1 ratio) was intravenously injected into vaccinated mice. After 24 hours, splenocytes were collected and analyzed by flow cytometry (h), and target cell killing was quantified (i). Data in a–d, g and i are shown as mean ± SD (n = 5). Statistical differences were calculated using one-way ANOVA with Tukey’s post hoc test.

Source data

Extended Data Fig. 3 Body weight change and survival curves.

a, Body weight change after mice were treated with various LNP vaccines. b, Mice survival curves after treatment with various LNP vaccines encapsulating OVA mRNA. c, Survival curves of mice receiving various LNPs encapsulating Pbk-Actn4 mRNA. Data in a are shown as mean ± SD (n = 8).

Source data

Extended Data Fig. 4 T cell immune responses elicited by C-a16 LNP encapsulating mRNA encoding SARS-CoV-2 spike protein.

Mice received intramuscular immunization with various LNP vaccines on days 0 and 21 (0.25 mg/kg). On day 35, splenocytes were collected and stimulated with SARS-CoV-2 RBD peptide pools. a and b, T cells were assessed for Th2 (IL-4, IL-5) and c, Th17 (IL-17a) intracellular cytokine expression. d, Evaluation of the expression of the cytotoxic marker CD107α expression in CD8+ T cells. Data are shown as mean ± SD (n = 5). Statistical differences were calculated using one-way ANOVA with Tukey’s post hoc test.

Source data

Extended Data Fig. 5 Evaluation of polyfunctional CD4+ and CD8+ T cells.

Mice received intramuscular immunization with various LNP vaccines on days 0 and 21 (0.25 mg/kg). On day 35, splenocytes were collected and stimulated with SARS-CoV-2 RBD peptide pools. CD4+ (a–g) and CD8+ (h–n) polyfunctional T cell percentages (%) were assessed. Data are shown as mean ± SD (n = 5). Statistical differences were calculated using one-way ANOVA with Tukey’s post hoc test.

Source data

Extended Data Fig. 6 Investigation of humoral immune responses and memory B cells elicited by various LNPs.

a, Mice received intramuscular immunization with various LNP vaccines on days 0 and 21 (0.25 mg/kg). IgG2c to IgG1 ratio was determined. Data are presented as mean ± s.d. b, Enumeration of RBD-specific B cells per spleen. Splenocytes were stimulated with SARS-CoV-2 RBD peptide pools and stained with various antibodies before flow cytometry analysis. c, Percentage of RBD-specific B cells categorized by germinal center (GC) or memory phenotype. GC B cells were defined as CD38−GL7+, and memory B cells were defined as CD38+GL7−. Data are shown as mean ± SD (n = 5). Statistical differences were calculated using one-way ANOVA with Tukey’s post hoc test.

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Supplementary information

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Gong, N., Kim, D., Alameh, MG. et al. Mannich reaction-based combinatorial libraries identify antioxidant ionizable lipids for mRNA delivery with reduced immunogenicity. Nat. Biomed. Eng (2025). https://doi.org/10.1038/s41551-025-01422-8

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